The rate of bone fractures in the United States is estimated at 6,000,000 individuals per year. In 1984 (Holbrock et al., 1984) these injuries resulted in a direct cost (i.e., excluding loss of income) of $17,000,000,000 per year. When a bone is completely fractured, a significant number of fractures require medical intervention beyond simple immobilization (casting), particularly those involving trauma. A major problem in such instances is the lack of proximity of the two bone ends (referred to as non-union). This results in an inappropriate and prolonged repair process, which may prevent recovery. The average length of time for the body to repair a fracture is 25-100 days, for moderate load-bearing, and one year for complete repair. Thus, both simple fractures and medically complicated breaks would benefit from novel therapeutic modalities which accelerate and/or complete the repair process. The same is true for those bone diseases (referred to as osteopenias) which result in a thinning of the bone the primary symptom of which is an often-debilitating fracture.
Primary Osteoporosis is an increased, progressive bone loss which accompanies the aging process. As such, it represents significant health risk in the United States which greater than 15 million Americans suffering from primary (idiopathic) osteoporosis resulting in a direct cost of $6,000,000,000 per year (Holbrock et al., 1984). Primary osteoporosis is the most common of the metabolic bone disease, and some 40,000-50,000 fracture-related deaths per year are attributed to this disorder. This mortality rate is greater than deaths due to cancer of the breast and uterus, combined. Significantly, this disorder, which is one of the osteopenias, is asymptomatic until a bone fracture occurs. Affected individuals typically fracture the radius, femoral head, or collapse vertebrae. Osteoporosis has a greater impact on the female population with larger numbers of women than men struck by this disorder, and a significant increase in the rate of osteoporosis occurs post-menopause. The rate of osteoporosis in these women is slowed but not ameliorated by estrogen replacement therapy. Indeed, there is no convincing medical evidence that any treatment is successful in restoring lost bone mass of any kind. Given the aging of the American population, patients with osteoporosis also represent a significant target population for effective and novel bone therapies.
There is no current treatment for lost bone mass, including various growth-promoting proteins and Vitamin D.sub.3. Likewise, there is no effective replacement or implant for non-union fractures or crush injuries of the bone. Currently, these latter types of injury utilize bovine (cow), or human cadaver bone which is chemically treated (to remove proteins) in order to prevent rejection. However, such bone implants, while mechanically important, are biologically dead (they do not contain bone-forming cells, growth factors, or other regulatory proteins). Thus, they do not greatly modulate the repair process. All of these concerns demonstrate a great need for new or novel forms of bone therapy.
Bone development results from the proliferation of mesenchymal cells, their differentiation into osteogenic progenitor cells, and the eventual calcification of cartilage and bone extracellular matrix (Urist et al., 1983). Human bone marrow contains a distinct cell population that expresses bone proteins and responds to growth factor .beta.1 (TGF-.beta.), but not to hematopoietic growth factors (Long et al., 1990).
Little information exists concerning the growth factors or cytokines controlling development of bone precursor cells (osteoprogenitor cells and preosteoblasts) into their differentiated progeny, the osteoblasts. Likewise, few studies address the impact of extracellular matrix (ECM) molecules on this stage of human bone cell development, or the impact of aging on either of these two areas. In the past, human bone cells (both precursor cells and osteoblasts) have been technically difficult to acquire and purification/characterization studies or protocols were few in number. Additionally, current in vitro models of bone formation are limited as the use of post-fetal mesenchymal tissue to generate bone cells often results in chondrogenesis, but is inadequate for osteogenesis, (Urist et al., 1983). Thus, information concerning the cellular activation signals, differentiation, and bone matrix production during the early phases of human bone cell development is limited, at best.
The regulation of chondro-osteogenic gene activation is induced during bone morphogenesis by an accumulation of extracellular and intracellular signals. (Urist et al., 1983). Importantly, extracellular signals are known to be transferred from both cytokines and extracellular matrix molecules (Urist et al., 1983), to responding cell surface receptor(s) resulting in eventual bone formation. The formation of bone occurs by two mechanisms. Direct development of bone from mesenchymal cells (referred to as intramembranous ossification; as observed in skull formation) occurs when mesenchymal cells directly differentiate into bone tissue. The second type of bone formation (the endochondral bone formation of skeletal bone) occurs via an intervening cartilage model.
The development and growth of long bones thus results from the proliferation of mesenchymal cells, their differentiation into osteogenic progenitor cells and (then) osteoblasts, cartilage deposition, and eventual calcification of the cartilage and/or bone matrix. Concurrently, bone is remodeled to form a tubular bone space in which hematopoietic cell differentiation occurs. Interestingly, the number of osteoprogenitor cells in adult bone seems too small to replace all of the large mass of bone normally remodeled in the process of aging of skeleton. (Urist et al., 1983). Our observations (vide infra) confirm this concept by showing that one (unexpected) source of osteoprogenitor cells is the bone marrow. This reduced progenitor cell number also implies that there is a disassociation of bone progenitor cell recruitment from subsequent osteogenic activation and bone deposition, and further suggests multiple levels of regulation in this process.
One of the central issues concerning bone formation regards the developmental lineages of the bone cell types, namely the osteoblast and the osteoclast. There is adequate evidence to suggest that osteoblasts arise from local mesenchymal cell populations, and that osteoclasts are derived from blood-born monocyte/macrophage cells. Fischman and Haye first demonstrated that monocytes fused to form osteoclasts in regenerating newt limbs (Fishman et al., 1962). Although the role of macrophage fusion remains controversial (Hattersley et al., 1989 and Horton et al., 1985), further evidence for the blood-born origin of the osteoclast was pioneered by LeDouarin using a chick:quail chimera in which nuclear morphology allows clear distinction of cell derivation. These studies conclusively demonstrated that osteoblasts and osteocytes are derived from the limb bud mesenchyma whereas osteoclasts arise from blood-born hematopoietic cells (Jotereau et al., 1978 and Le Douarin, 1973). The importance of these observations was subsequently shown by the successful cure (by osteoclasts) of osteopetrosis utilizing bone marrow transplantation in both animals (Ash et al, 1980), and humans (Coccia et al, 1980). While such data conclusively show the hematogenous origin of the osteoclast, little knowledge exists on the nature or location of the stem cell population(s) capable of differentiating into bone-forming osteoblasts.
Like other developing tissues, bone responds to bone-specific, and other soluble growth factors. TGF-.beta. is a member of a family of polypeptide growth regulators which affects cell growth and differentiation during developmental processes such as embryogenesis and tissue repair (Sporn et al., 1985). TGF-.beta. strongly inhibits proliferation of normal and tumor-derived epithelial cells, blocks adipogenesis, myogenesis, and hematopoiesis (Sporn et al., 1985). However, in bone, TGF-.beta. is a positive regulator. TGF-.beta. is localized in active centers of bone differentiation (cartilage canals and osteocytes) (J. Massague, 1987), and TGF-.beta. is found in high quantity in bone--suggesting that bone contains the greatest total amount of TGF-.beta. (J. Massague, 1987 and Gehron Robey et al., 1987. During bone formation, TGF-.beta. also promotes chondrogenesis (J. Massague, 1987)--an effect presumably related to its ability to stimulate the deposition of extracellular matrix (ECM) components (Ignotz et al., 1986). Besides stimulating cartilage formation, TGF-.beta. is synthesized and secreted in bone cell cultures, and stimulates the growth of sub-confluent layers of fetal bovine bone cells, thus showing it to be an autocrine regulator of bone cell development (Sporn et al., 1985).
In addition to TGF-.beta., other growth factors or cytokines are implicated in bone development. Urist and co-workers have been able to isolate various regulatory proteins which function in both in vivo and in vitro models (Urist et al., 1983). Bone morphogenic protein (BMP), originally an extract of demineralized human bone matrix, has now been cloned (Wozney et al., 1988), and when implanted in vivo results in a sequence of events leading to functional bone formation (Wozney et al., 1988 and Muthukumaran et al., 1985). The implanting of BMP is followed by mesenchymal cell migration to the area of the implant, differentiation into bone progenitor cells, deposition of new bone, and subsequent bone remodeling to allow the establishment of bone marrow (Muthukmaran et al., 1985). A number of additional growth factors exist which regulate bone development. In particular, bone-derived growth factors (BDGF) stimulate bone cells to proliferate in serum-free media. (Hanamura et al., 1980 and Linkhart et al., 1986). However, these factors seem to function at a different level from BMP (Urist et al., 1983).
The extracellular matris (ECM) varies in its tissue composition throughout the body, consisting of various components such as collagen, proteoglycan, and glycoprotein. (Wicha et al., 1982). Numerous studies point to the influences of ECM in promoting cellular development. Gospodarowicz et al. demonstrated that ECM, the natural substrate surrounding cells in vivo, greatly affects corneal epithelial cell proliferation in vitro (D. Gospodarowicz and C Ill, 1980 and Gospodaroiwcz et al., 1980). Studies by Reh et al. show that extracellular components such as laminin are involved in inductive interactions which give rise to retinal and retinal pigmented endothelium (Reh et al., 1987). Also, differentiation and growth of mammary epithelial cells are profoundly influenced by ECM components, and mammary cell growth in vivo and in vitro requires type IV collagen (Wicha et al., 1982), Finally, studies from one of the inventor's laboratories show that bone marrow ECM also plays a major role in hematopoiesis in that complex ECM extracts greatly augment cell proliferation (Campbell et al., 1985), and that marrow-derived ECM contains specific cytoadhesion molecules (Campbell et al., 1987; Campbell et al., 1990; M. W. Long and V. M. Dixit, 1990, Long et al., 1990; and Long et al., 1992).
A number of non-collagenous matrix proteins, isolated from demineralized bone, are involved in bone formation. Osteonectin is a 32 kDa protein which, binding to calcium, hydroxyapatite and collagen, is felt to initiate nucleation during the mineral phase of bone deposition. (Termine et al., 1981). In vivo analysis of osteonectin message reveals its presence in a variety of developing tissues (Nomura et al., 1988 and Holland et al., 1987). However, it is present in its highest levels in bones of the axial skeleton, skull, and the blood platelet (megakaryocyte) (Nomura et al., 1988). Bone gla-protein (BGP, osteocalcin) is a vitamin K-dependent, 5700 Da calcium binding bone protein which is specific for bone and may regulate Ca++ deposition (Termine et al., 1981; Price et al., 1976, and Price et al., 1981). Other bone proteins seem to function as cytoadhesion molecules (Oldberg et al., 1981 and Somerman et al., 1987), or have unresolved functions (Reddi, 1981).
While bone morphogenesis is ECM dependent, bone ECM also contains a number of the more common mesenchymal growth factors such as PDGF, basic, and acidic fibroblast growth factor (Urist et al., 1983; Linkhart et al., 1986; Hauschka et al., 1986; and Canalis et al., 1985). These activities are capable of stimulating the proliferation of mesenchymal target cells (BALB/c 3T3 fibroblasts, capillary endothelial cells, and rat fetal osteoblasts). As well, bone-specific proliferating activities such as the BMP exist in bone ECM. While these general and specific growth factors undoubtedly play a role in bone formation, little is understood concerning the direct inductive/permissive capacity of bone-ECM or bone proteins themselves on human bone cells or their progenitors. Nor is the role of bone ECM in presenting growth factors understood--such "matricrine" (factor:ECM) interactions may be of fundamental importance in bone cell development.
The present invention provides the isolation, purification, characterization, and growth factor responsiveness of these precursors to osteoblasts, and the identification of a human osteoprogenitor cell. Immunological separation of bone marrow non-adherent low-density (NALD) cells results in a marked enrichment of bone precursor cells that express osteocalcin, osteonectin, and bone alkaline phosphatase. Flow cytometric analyses show that distinct cell subpopulations exist among these isolated cells. The majority of the bone antigen-positive cells are approximately the size of a lymphocyte, whereas other, antibody-separated subpopulations consist of osteoblasts and osteoprogenitor cells. In serum-free cultures, TGF-.beta. stimulates the small, antigen-positive cells to become osteoblasts as these cells both increase in size, cellular complexity, and express increased levels of osteocalcin and alkaine phosphatase.
Antibody-separated cells also contain a separate population of progenitor cells that form colonies of osteoblast cells when cultured in serum-free, semi-solid media. Two types of osteoprogenitor cells are observed: a colony-forming cell (CFC) that generates several hundred bone antigen-positive cells, and a more mature cluster-forming cell that has a lesser proliferative potential and thus generates clusters of 20-50 antigen-positive cells. Osteopoietic colony-forming cells and cluster-forming cells have an obligate, but differential requirement for osteogenic growth factors. The CFCs respond to TGF-.beta., basic fibroblast growth factor (bFGF), bone morphogenetic protein-2 BMP-2), and 1, 25-dihydroxy vitamin D3 (1,25-OH D3). In contrast to the colony-forming cells, cluster-forming cells are regulated predominately by 1,25-OH D3 and TGF-.beta., but fail to respond to bFGF. We conclude that human bone marrow contains a non-hematogenous, heterogeneous population of bone precursor cells among which exists a population of proliferating osteoprogenitor cells. The use of these bone precursor cell populations should yield important information on their role in osteogenesis in both health and disease.